Process for preparing higher fatty acid salts of neomycin



3,022,286 PROCESS FOR PREPARING HIGHER FATTY ACID SALTS F NEOMYCINGerald H. van de Griendt, Summit, N.J., assignor to S. B. Penick andCompany, New York, N.Y., a corporation of Delaware No Drawing. FiledDec. 14, 1959, Ser. No. 859,160 8 Claims. (Cl. 260-210) This inventionrelates to compositions of matter known in the art of chemistry asorganic salts of neomycin and more particularly to processes for makingsuch compositions.

The invention here is described as residing in the concept of dissolvingapproximately equivalent quantities of 3,iPZZ,Z$$ Patented Feb. 20, 1962ice no apparent skin irritation. Thus the higher fatty acid neomycinbase with the appropriate higher fatty acid in a lower alkanol solventand at a final pH between 6.5- 7.5 to form a chemical compound in whichneomycin is, associated with a higherfatty acid in the form of a truesalt.

As used herein the term higher fatty acid means an aliphaticmonocarboxylic acid containing 8 to 18 carbon atoms. -The aliphaticportion of the acid may be straight chain or branched and saturated orunsaturated. The better known higher fatty acids are straight chain,saturated compounds containing 8 to 18 carbon atoms; the acidsparticularly useful in the practice of my invention can also berecognized as those whose alkali metal salts are generally known assoaps.- My invention, however, is not so limited and contemplates allaliphatic fatty acids having 8 to 18 carbon atoms and which producewaterinsoluble salts of neomycin. And as used herein the term loweralkanol means saturated alcohols having less than 3 carbon atoms, i.e.,methyl and ethyl alcohols.

Neomycin, also referred to as neomycin base, is a naturally occurringantibiotic elaborated by a soil organism of the Streptomyces genus whencultured on the appropriate nutrient media. The neomycinthus produced isa basic, water-soluble compound active against many gram positive andgram negative micro-organisms. The antibiotic is often isolated andincorporated into pharmaceutical preparations in the form of its mineralacid salts, most commonly neomycin sulfate and chloride. Because of thevery high water solubility of neomycin mineral acid salts (one gram ofneomycin sulfate dissolves in one milliliter of water), these salts arereadily leached from preparations containing them. Hence, the beneficialeffects of neomycin are not fully realized in such preparations whentopically applied.

The higher fatty acid salts of neomycin, unlike neomyein base orneomycin mineral acid salts, are waxlike and practically insoluble inwater and thus may be more effectively utilized in ointments, lotions,creams, soap bases and other preparations designed for topical use.Replacement of the mineral acid anion by a higher fatty acid anion doesnot materially detract from the germicidal properties of the neomycinmoiety. When neomycin sulfate and neomycin palmitate were compared intheir anti-fungal and anti-bacterial properties against 34representative micro-organisms including C. diphtheria, S. typhosa, St.faecolis, Candida albicans, Trichophytin tonsurans and Rizopusnigricans, the fatty acid salt was at least as effective as the sulfate;and in the case of 23 of the organisms tested, even smallerconcentrations of the palmitate were sufiicient to produce equivalentinhibition in the growth of the test bacteria and fungi.

salts of neomycin can be safely and effectively utilized in topicalpreparations for the treatment of dermatological conditions caused byneomycin susceptible organisms. These salts can also be incorporatedinto compositions su'ch as hand creams which are used to obtainsubstanunsuited for large scale production.

tially antiseptic conditions. In many cases, the salts have the addedadvantage of producing their own emolient or demulcent effect.

' The mere mixing of higher fatty acids with neomycin base in aqueoussolution has not proven to be a satisfactory method for the preparationof fatty acid salts of neomycin. The reaction is hampered by the poorsolubility of both the reactant fatty acid and the resultant fatty acidsalt. Particles of the poorly soluble acid became coated with the evenmore insoluble salt giving a system from which it is extremely difficultto isolate the low yields of product obtained. Such a process is grosslyIf it be desired to operate in an aqueous system, one must utilize thereaction of a mineral acid salt of neomycin with an alkali metal salt ofthe reactant fatty acid. This approach is not completely satisfactorybecause of the fact that one of the reactants is a soap and the productis soap-like and, hence difficult to handle, isolate cleanly or purify.Drying may also be a problem. Inorganic salts such as sodium sulfate,potassium chloride and potassium sulfate are; obtained as by-productsand may contaminate the product.

I have discovered that neomycin reacts in alcoholic solution and undercarefully controlled conditions with the appropriate higher fatty acidto give excellent yields of the desired neomycin fatty acid salt. Aswill be discussed below, the pH and the ratio of reactants used must becarefully controlled in order to obtain good yields of high gradeproduct. Deviation from the optimum conditions will still give the fattyacid salt of neomycin but in a lessened degree of purity.

It is therefore; an object of the present invention to provide aconvenient method for the preparation of higher fatty acid salts ofneomycin free of inorganic and other contaminants.

The reaction of neomycin base with higher fatty acids is effected,according to my process, in a lower alkanol, preferably methanol orethanol. Mixtures of those alcohols are also satisfactory. It is notnecessary that the alcohol used be anhydrous; the usually availablecommercial grades of methyl or ethyl alcohol are most often employed.The order of mixing the reactants is not critical and all methods ofmixing the reactants with each other and with the solvent areequivalent. Usually a solution of the acid is added to a solution of theneomycin base but the reverse order may be employed. The fatty acid canbe added directly to the alcoholic solution of the neomycin base.Temperature control is not required and the process is carried out atambient temperatures. A certain amount of warming results when thereactants are mixed raising the temperature of the reaction mixture fromroom temperature to about 40-45 degree Centigrade. Slight warming may beutilized to further solubility; this is especially the case when it isdesired to clarify the solution with charcoal before recovery of theproduct.

In order to achieve consistently high yields of superior product, it iscritical that the final pH of the reaction mixture and the ratio ofreactants fall within the limits stated below. The final pH of thereaction mixture must be about 6.5 to 7.5. If the final pH is allowedtovary from the suggested range, either the fatty acid will precipitatefrom solution or certain other salts of neomycin will be found inconjunction with the desired neomycin fatty acid salt. While it is notan object of the present invention to prepare such compositions, theycan be made by varying the general procedure of my process. However, thebest way to obtain the proper final pH and pure product is to titratethe neomycin base in alcohol solution with the fatty acid dissolved inthe same solvent or to add the calculated approximate stoichiometricamount of acid (directly or in solution) to a solution of the base.

A slight stoichiometric excess of acid is not harmful and in some casesmay even be helpful; but the yield and quality of the product, asmeasured by its biological potency, fall off sharply at even slightlyalkaline pI-Is in excess of 7.5. If necessary, the H of the reactionmixture may also be adjusted by the addition of small amounts of mineralacid or inorganic base. Other variations will be obvious to one skilledin the art.

The higher fatty acid salts of neomycin have a desirable waxyconsistency. The palmitate and higher salts are sufiiciently insolublein alcoholic solution so as to be capable of isolation directly byfiltration, centrifugation, etc. Chilling, although not essential, maybe utilized to diminish the solubility of the product. The presence ofsmall amounts of water in the alcoholic solvent will accomplish the sameresult and may even be added for that purpose. The lower salts are toosoluble to be isolated directly and are recovered by evaporation of thesolvent, preferably by spray drying. This technique can be usedsuccessfully because of the absence of contaminating byproducts; thepresence of small amounts of water will not interfere. In certainsituations, the presence of up to about 20 percent of water in thealcoholic solvent used will not be detrimental and can be tolerated.

The novel neomycin higher fatty acid salts of my invention have beenshown by X-ray analysis to be true salts and not mere mixtures ofneomycin with the reactant fatty acid.

he scope and utility of my invention is further illustrated by thefollowing examples:

Example I This example illustrates the preparation of neomycin palmitatefrom neomycin base and palmitic acid in alcohol solution. In a smallerscale run, 80 grams of palmitic acid was added to methanolic solutioncontaining 20 grams of neomycin base. Sufficient methanol was added toproduce a clear solution at 50-55 degrees Centigrade. The final solutioncontained about 500 milliliters of methanol and had a pH of 6.8. Afterchilling to ice temperature, 95 grams of neomycin palmitate having apotency of 205 mcg./ mg. was obtained on filtration.

In a larger scale run, a solution of 7-8 percent of neomycin base in22-24 gallons of methyl alcohol was used as the starting material. Thissolution was obtained by the methanol extraction of crude neomycin.Palmitic acid, amounting to about three times the weight of neomycinbase solids present, was added to the neomycin the first agar plate 50mcg./ml.

extract, and the temperature raised to effect complete solution (about45-50 degrees centrigrade). The clear solution (pH of about 7) wascooled to ice temperature and the resultant solid collected byfiltration. There was obtained about 20 kilograms of neomycin palmitatehaving a potency of about 200 rncg./mg. The product was an off-whitepowder soluble in methanol, sparingly soluble in ethanol, slightlysoluble in propylene glycol and insoluble in glycerin or water.

Example 11 This example illustrates the preparation of neomycinpelargonate from neomycin base and pelargonic acid. Using the generalprocedure of the first example, the weight ratio of reactants was about2.1 parts of acid to 1 part of neomycin base. The final solution had apH of about 7 also but the product was too soluble to recover byfiltration. A quantitative yield of neomycin pelargonate was obtained byspray drying the reaction mixture. The product was an off-white powdersoluble in methyl alcohol but insoluble in water.

Example III This example illustrates the preparation of neomycinundecylenate from neomycin base and undecylenic acid. A solution wasprepared containing 277 grams of neomycin base in 7740 milliliters ofmethanol. After the addition of about 550 grams of undecylenic acid, thesolution had a final pH of 7.1. Spray drying gave 640 grams of neomycinundecylenate having a potency of 250 meg/mg. The product was anoff-white powder soluble in methyl alcohol, ethyl alcohol or propyleneglycol and practically insoluble in glycerin or water.

Example IV This example illustrates the preparation of neomycincaprylate from neomycin base and caprylic acid. Using the generalprocedure of Example III, 446 grams of caprylic acid was added to asolution of 323 grams of neomycin base in 9020 milliliters of methanol.The final reaction mixture (pH about 7.1) was spray dried to yield 610grams of neomycin caprylate having a potency of 405 meg/kg. The productwas an off-white powder soluble in methyl alcohol and insoluble inwater.

The germicidal properties of my novel neomycin fatty acid salts wereillustrated by the comparison of a typical compound, neomycin palmitate,with neomycin sulfate. The general procedure is described as follows:Neomycin sulfate was dissolved in pH 8.0 bufiier to a concentration of1000 meg/mg. and neomycin palmitate was dissolved in absolute ethanol tothe same activity. Each solution was sterilized by filtration throughsterile glass filters. The anti-bacterial sensitivity tests wereperformed using a two-fold serial dilution tube technique for allcultures except Hemophilus influenza and pertussis which were done bythe plate dilution method. The final concentration of the antibioticswere adjusted so that the first tube in the dilution series containedmeg/ml. and For the bacterial tube sensitivities, trypticase soy brothwas used for Strep. virz'dans and Cl. perfrigens where thioglycollatebroth was employed. The antifungal activity of the drugs were determinedusing Sabourauds broth. Bordet-Gengou agar plus 15 percent rabbit bloodwas used for the plate sensitivities. All cultures were obtained fromthe American Type Culture Collection. The 24 hour broth cultures werediluted 1:1000 in broth prior to their addition to the serially diluteddings but those cultures which grew profusely were diluted l:10,000. Allbacterial tube and plate sensitivities were incubated at 37 degreescentigrade for 18 hours while the antifungal sensitivities wereincubated at 25 degrees centrigrade for 72 hours. The minimal inhibitoryconcentration of the test compounds were recorded as the lowestconcentration in meg/ml. necessary to inhibit growth.

Test Bacterial Organism Minimal Inhibitory Concentration Species CulturePalmi- Sulfate No. tate A. aerogenes 100 1. 56 3.12 C. diphtheriae. 3110.10 0.10 S. ty hosa- 401 3.12 12. 5 E. 002' 536 6. 25 12. 5 H.influenzae. 554 25.0 6. 25 H. pertussis-.. 558 6. 25 1. 56 K. pneumoma602 1. 56 3.12 Pr. vulgaris.-- 720 25.0 6. 25 Pr aeruginosa 813 12.5 12.5 S. schott. (Para 010 6. 25 25.0 Sh. paradysenteriae 959 25.0 12. 5 Mpz ogenes (albus) 1200 0.78 0.78 M pyogenes (aureus) 1209 1. 56 3.12 A lpyogenes (citreua)... 1206 3.12 6.25 M. pyagenes (epidermidzs) 1216 1.56 0. 39 St. viridans 1300 3.12 100 St. hemlyt1cus- 1307-3 0. 78 6. 25St. Pyoljenes. 13074 0.20 1. 56 St. faecalis 1341 50 100 D. pneumomue.1900 1. 56 12. 5 Cl. perjringens 1054. 3 3.12 250 Test Fungal OrganismMinimal Inhibitory Concentration Species Culture Palmi- Sulfate No. tateCandida albicans M63 500 500 C'ryptococcus neojormam. M260 500 500Microsporum adom'ni- M80 15. 6 125 Microsporum gy scum. M82 15.6 500Sporotrichum so enckii. 17161 62.5 500 Trz'chophyton mentagrop 1730231.2 250 Trichophyto'n rubrum- 62. 5 500 500 500 15. 6 500 62.5 500 31.2 500 62.5 500 62. 5 500 In summary, it can be said that the higherfatty acid salts of neomycin are white solids practically insoluble inwater. They possess a waxy consistency and germicidal properties whichrender them useful in pharmaceutical compounding especially inpreparations designed for topical application. These salts areconveniently prepared by the reaction of neomycin base and theappropriate fatty 6 acid in lower alkanol solution. In addition togiving high yields of good quality product, the process has the addedadvantage of being able to utilize alcoholic extracts of neomycin basedirectly as obtained from Streptomyces fermentation beers.

Having described my invention, I claim:

1. The process for preparing higher fatty acid salts of neomycin whichcomprises: dissolving approximately stoichiornetrically equivalentquantities of neomyciu base and a higher fatty acid in a lower alkanolso that the final pH of the reaction mixture is between about 6.5 andabout 7.5.

2. The process for preparing higher fatty acid salts of neomycin whichcomprises dissolving approximately stoichiometrically equivalentqu'atities of neomycin base and a higher fatty acid in a lower alkanolso that the final pH of the reaction mixture is between about 6.5 andabout 7.5 and recovering the higher fatty acid salt of ncomycin thusformed from the reaction mixture.

3. The process according to claim 2 wherein the fatty acid isundecylenic acid.

4. The process according to claim 2 wherein the fatty acid is pelargonicacid.

5. The process according to claim 2 wherein the fatty acid is caprylicacid.

6. The process according to claim 2 wherein the higher fatty acid ispresent in slight excess of the stoichiometric quantity.

7. The process according to claim 2 wherein the lower alcoholic solventcontains up to 20 percent of water.

8. The process for preparing IWOIHYClIl palmitate which comprisesdissolving approximately stoichiornetrically equivalent quantities ofneomycin base and palmitic acid in methyl alcohol at a final pH of about7 and recovering the neomycin palmitate thus formed from the reactionmixture.

References Cited in the file of this patent UNITED STATES PATENTS2,916,483 Dutcher Dec. 8, 1959 OTHER REFERENCES Baker-Drug and CosmeticInd., 80 (1957), 458 and 552-3, cited in C.A., vol. 51 (1957), 12438H.

1. THE PROCESS FOR PREPARING HIGHER FATTY ACIDS SALTS OF NEOMYCIN WHICHCOMPRISES: DISSOLVING APPROXIMATELY STOICHIOMETRICALLY EQUIVALENTQUANTITIES OF NEOMYCIN BASE AND A HIGHER FATTY ACID IN A LOWER ALKANOLSO THAT THE FINAL PH OF THE REACTION MIXTURE IS BETWEEN ABOUT 6.5 ANDABOUT 7.5.